These investigations are directed towards: (1) identifying and locating along the collagen molecule the key borohydride-reducible and non-reducible lysyl-derived cross-links involved in fiber and bundle organization; (2) determining hormonal effects on these cross-links. The ultimate objective is to understand the basis for sex differences in vascular aging and focal atherogenesis in man. During the initial grant year, a definitive report was made on sequence of appearance of NaB3H4-reducible collagen cross-links as fibers and bundles appear in electron micrographs of connective tissue developing around the implanted Ivalon sponge in adult male rats (Biochem. J. 177, 1979). Of the reducible collagen cross-links, non-glycosylated dihydroxylysinonorleucine (DHLNL) undergoes the greatest percentage increase. The changing relationship between amounts of reducible hydroxylysinonorleucine, diglycosylated and non-glycosylated DHLNL suggests three post-translational steps involving lysyl hydroxylase and lysyl oxidase during fiber and bundle formation. Additional progress includes introduction of precursor radioactive lysine into collagen cross-links as fibers and bundles in the Ivalon implant system. Currently, a search is on for previously unrecognized non-reducible lysyl-derived collagen cross-links in developing collagen. Finally, work has started on identifying the locus of the only known non-reducible collagen cross-link, hydroxyaldolhistidine (HAH). HAH-peptides from steer skin collagen are isolated and subjected to a series of organic chemical reactions yielding the three peptides that contribute their prosthetic groups to the cross-link. Amino acid sequences of these peptides will be determined to identify peptide position along the course of the alpha (and possibly alpha2) chain of collagen.